The metal binding and activity of protein Mn and Fe sites is essential to life, but its structural basis is incompletely understood. This project contributes to the Program by pursuing integrated mutational and structural studies of the Mn and Fe binding sites in Mn and Fe superoxide dismutases (SOD) and the ribonucleotide reductase R2 subunit (RNR R2), and by designing such sites into green fluorescent protein (GFP). The Fe and Mn SODs have identical folds and active site geometries, but the SOD activity of these enzymes is dependent on specific metal binding. Replacement of second and third shell residues of MnSOD will be used to characterize the structural basis for the activity, geometry, and specificity of the metal-binding active site. The halophilic, thermophilic E. halophila MnSOD will be used as a template for probing more extreme mutations than those possible with the human enzyme. In parallel, we will study the di-iron binding site of RNR R2 to understand the effects of ligand stereochemistry, ligand neighborhood, and protein environment on this alternative mode of Fe and Mn binding by proteins. Aided by the Program Core modules, the DEZYMER design algorithm, and the Quantitative Metalloprotein Database, knowledge gained from the SOD and RNR R2 systems will be applied to design Mn and Fe sites in the GFP system. The screening capabilities of the GFP system provide a powerful method for optimizing metal geometry and activity and complement structure-based design efforts. By transplanting the Mn and Fe site templates from MnSOD and FeSOD into the GFP environment, we will test and improve our understanding of metal site structure/function relationships. Our common approach with all 7 Projects within this Program is to employ recursive design to alter and transplant metal sites followed by structural and functional characterization. Thus our studies and results aim to contribute to all 7 Projects, whose common goal is to discover the factors controlling metal site chemistry, structure, and activity, including domain architecture, ligand type and geometry, non-covalent interactions and conformation change. Knowledge of the relationship between protein structure and metal site activity in the SOD and RNR systems is crucial for understanding the biology of these medically relevant enzymes, which are critical in cellular defense against oxidative stress and DNA replication, respectively.